PKMYT1 inhibitor and application thereof

Abstract

The invention relates to an amine derivative with PKMYT1 inhibitory activity as shown in a formula I, and an isotope form, a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a pharmaceutically acceptable solvate, a hydrate, a prodrug and a polymorphic substance of the amine derivative, and describes a preparation method of the amine derivative and application of the amine derivative in preparation of medicines for treating diseases related to PKMYT1 activity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a compound capable of inhibiting myelin transcription factor 1 kinase, a preparation method and a composition thereof, and use of the compound in preparing medicines. Background Art

[0002] Myelin transcription factor 1 kinase PKMYT1 is also known as MYT1. The gene is located on chromosome 16p13.3 and encodes a protein membrane-associated tyrosine and threonine-specific CDC2 inhibitory kinase. The encoded protein is mainly located in the endoplasmic reticulum and Golgi complex. PKMYT1 negatively regulates the cell cycle mainly by phosphorylating Tyr15 and Thr14 of cyclin-dependent kinase 1 (Cdk1). PKMYT1 belongs to the WEE1 kinase family, which consists of three serine / threonine kinases that share a conserved molecular structure, including WEE1, PKMYT1 and WEE1B. Among them, WEE1 and PKMYT1 play a major role in the entry of cells into dominant division. The difference between WEE1 and PKMYT1 is that WEE1 phosphorylates Cdk1 on Tyr15 and participates in the regulation of CDK1 / 2 activity, while PKMYT1 phosphorylates Cdk1 on Tyr15 and Thr14.

[0003] It is well known that in the early stage of the cell cycle, Cdc2 protein kinase is inactive. As cells enter the S phase, cyclin B accumulates and forms a complex with Cdc2. The complex is also inactive, but under the action of kinases including WEE1 and MYT1, it can phosphorylate Tyr15, Thr14 and Thr161. At this time, the complex remains inactive. Then a specific phosphatase (Cdc25) removes the phosphorylation of Thr14 and Tyr15, produces an active Cdc2 / Cyclin B complex, initiates downstream signals, and causes cells to enter mitosis. Related studies have shown that the inhibition of Cdc2 activity is critical for the time when cells enter mitosis, and its premature activation can lead to mitotic catastrophe and cell death. Inhibition of PKMYT1 may lead to premature activation of Cdc2, thereby killing rapidly proliferating cells.

[0004] PKMYT1 is often overexpressed in solid tumors and blood tumors, including clear cell renal cell carcinoma and hepatocellular carcinoma. Related studies believe that PKMYT1 is crucial in many tumors. Studies have found that PKMYT1 downregulation has a weak effect in cells without DNA damage, but plays a very important role in cells with DNA damage. In cells with DNA damage, the loss of PKMYT1 function is particularly sensitive because these damaged cells will be more likely to enter mitosis prematurely, which will cause damage to genetic chromosomes and induce mitotic chaos. This also further shows that PKMYT1 inhibitors may become a method for treating tumors.

[0005] Clinical studies have found that CCNE1 gene amplification on chromosome 19q12 is common in many tumor types, including breast cancer, endometrial cancer, gastric cancer, and ovarian cancer, accounting for about 5-40%. The CCNE1 gene encodes cyclin E1 (cyclin E). Cyclin E is mainly involved in the transition from the G1 phase to the S phase of the cell cycle. After entering the late G1 phase of the cell cycle, it can bind to cyclin-dependent kinase 2 (CdK2), activate E2F transcription factor and enter the S phase. The expression level of cyclin E is strictly controlled in the normal cell cycle. Its high level of expression is associated with genomic instability, whole genome doubling, and chemotherapy-related drug resistance, which makes cancers containing CCNE1 gene amplification an important unmet clinical need. Although corresponding CDK2 inhibitors have entered clinical research, therapeutic drugs to solve this problem are still very important.

[0006] Studies have shown that in healthy cells, cyclin B-CdK1-related levels increase in the cytoplasm during the G2 phase of the cell cycle, and PKMYT1 can limit the activation and nuclear translocation of CDK1 until the onset of mitosis. In pathological conditions, when CCNE1 is amplified or overexpressed, cells suffer replication stress, and cyclinB-CdK1 levels increase early in the cell cycle from enhanced MMB-FOXM1 transcription. At this time, PKMYT1 is essential for limiting CDK1 activity until DNA replication is completed and mitosis begins. In CCNE1-amplified cells, PKMYT1 inhibition or gene knockout causes CDK1 to be activated before DNA replication is completed, and then enters mitosis prematurely, leading to catastrophic DNA damage and cell death. Corresponding preclinical studies have revealed that PKMYT1 kinase inhibition is synthetically lethal to CCNE1 amplified genes. This suggests that PKMYT1 kinase inhibitors may become a treatment for CCNE1 gene amplified tumors.

[0007] There have been a lot of studies on PKMYT1 inhibitors, but most products have not performed well in animal models due to poor pharmacokinetics and weak bioavailability. For example, the reported clinical candidate drug RP6306 only showed 37% bioavailability and low exposure (J. Szychowski et al. J. Med. Chem. 2022, 65, 10251-10284). Summary of the invention

[0008] The purpose of the present invention is to provide a compound having PKMYT1 inhibitory activity and application thereof.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] The first aspect of the present invention provides a compound of formula I or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0011]

[0012] in,

[0013] X, Y, and Z are independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl;

[0014] Or, Y, X and / or Z and the connected carbon atoms form a cyclic structure, wherein the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring, and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O;

[0015] U is N or C. When U is N, X does not exist.

[0016] W is N or CR 1 , where R 1 is selected from hydrogen, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 One of cycloalkyl, aryl, heteroaryl, alkenyl, and alkynyl;

[0017] G1 and G2 are independently selected from one of hydrogen, deuterium and fluorine.

[0018] The above substitution may be one or more hydrogens replaced by a substituent, and the substituent may be selected from deuterium, halogen, C 2-6 One or more of the alkyl groups.

[0019] Further, the compound is a compound represented by Formula Ia or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0020]

[0021] Wherein, X, Y, and Z are independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl.

[0022] Further, the compound is a compound represented by Formula Ib or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0023]

[0024] Wherein, X is selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl;

[0025] Y and Z and the connected carbon atoms form a cyclic structure, wherein the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring, and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O.

[0026] Further, the compound is a compound represented by Formula Ic or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0027]

[0028] wherein Z is selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl;

[0029] Y, X and the connected carbon atoms form a cyclic structure, wherein the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O.

[0030] Further, the compound is a compound represented by formula Id or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0031]

[0032] Wherein, Y and Z are selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl.

[0033] Further, the compound is a compound represented by Formula Ie or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph,

[0034]

[0035] Further, X, Y, and Z are independently selected from one of hydrogen, chlorine, fluorine, cyano, methyl, ethyl, propyl, fluoromethyl, trifluoromethyl, and deuterated methyl; or,

[0036] Y, X and / or Z and the connected carbon atoms form one of an alicyclic ring, an aromatic heterocyclic ring, or an alicyclic heterocyclic ring, for example, C 3-10 Alicyclic, C 3-10 Aromatic heterocyclic ring, C 3-10 Aliphatic heterocycle.

[0037] Furthermore, the compound represented by formula I is a compound represented by the following structural formulas I-1 to I-50:

[0038]

[0039]

[0040] The second aspect of the present invention provides a pharmaceutical composition, comprising one or more of the compound represented by formula I described in the first aspect, its isotopic form, stereoisomers, tautomers, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, hydrates, prodrugs and polymorphs.

[0041] Furthermore, in the pharmaceutical composition, the compound represented by any of the above formula I or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph is used in combination with other drugs.

[0042] The third aspect of the present invention provides a use of the compound represented by formula I described in the first aspect or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph or the pharmaceutical composition described in the second aspect in the preparation of a PKMYT1 inhibitor.

[0043] Furthermore, the PKMYT1 inhibitor is administered orally, parenterally, intravenously or transdermally.

[0044] The fourth aspect of the present invention provides a use of the compound of formula I described in the first aspect or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph or the pharmaceutical composition described in the second aspect in the preparation of a drug for treating diseases mediated by PKMYT1 activity.

[0045] Furthermore, the PKMYT1 activity-mediated conditions include, but are not limited to, solid tumors, blood tumors, and DNA damage response cancers, wherein the DNA damage response cancers include, but are not limited to, breast cancer, endometrial cancer, gastric cancer, ovarian cancer, and prostate cancer.

[0046] Furthermore, the drug is administered orally, parenterally, intravenously or transdermally.

[0047] Furthermore, in the drug, the compound represented by any one of the above formula I or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph is used in combination with other drugs.

[0048] As used herein, the following definitions and terms shall apply unless otherwise indicated.

[0049] "R" and "S", as terms for describing isomers, are descriptors of the stereochemical configuration of an asymmetrically substituted carbon atom. The designation of an asymmetrically substituted carbon atom as "R" or "S" is accomplished by applying the Cahn-Ingold-Prelog priority rules, which are well known to those skilled in the art and are described in Section E, Stereochemistry, of the International Union of Pure and Applied Chemistry (lUPAC) Rules of Organic Nomenclature.

[0050] The term C used here i-j means that the part has ij carbon atoms. For example, "C 1-10 "Alkyl" means an alkyl unit having any number of carbon atoms between 1 and 10.

[0051] As used herein, "alkyl" refers to a fully saturated straight chain, branched alkane radical. In certain embodiments, the alkyl contains 1-6 carbon atoms. In certain embodiments, the alkyl contains 1-4 carbon atoms. In certain embodiments, the alkyl contains 1-3 carbon atoms. In other embodiments, the alkyl contains 2-3 carbon atoms, and in other embodiments, the alkyl contains 1-2 carbon atoms. The non-limiting examples of exemplary alkyls include yl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-heptyl, n-octyl, etc. In addition, the term "cycloalkyl" refers to a monocyclic or bicyclic saturated carbocyclic ring, each ring having 3 to 10 carbon atoms.

[0052] The term "alkenyl" refers to a carbon chain containing at least one carbon-carbon double bond, which may be linear or branched, or a combination thereof. 2-5 Alkenyl includes ethenyl, propenyl, 2-methyl-1-propenyl, etc.; the term "alkynyl" refers to a carbon chain containing at least one carbon-carbon triple bond, which may be straight chain or branched chain, or a combination thereof. 2-3 Examples of alkenyl groups include alkynyl or propargyl.

[0053] The term "alicyclic ring" refers to a carbon ring connected by 3 or more carbon atoms, and the two adjacent carbon atoms in the ring may be single-bonded, double-bonded or triple-bonded, and the number of rings may be one or more; the term "alicyclic heterocyclic ring" refers to at least one carbon atom in the alicyclic ring replaced by a heteroatom, and the heteroatom may be N, O, S, etc., such as ethylene oxide, tetrahydrofuran, dioxane, etc. The term "aromatic heterocyclic ring" refers to an aromatic ring having one or more heteroatoms (e.g., oxygen, nitrogen or sulfur), such as furan, thiophene, pyrrole, pyridine, pyrimidine, quinoline, etc. The alicyclic ring described in the present invention includes substituted or unsubstituted alicyclic rings, the alicyclic heterocyclic ring includes substituted or unsubstituted alicyclic heterocyclic rings, and the aromatic heterocyclic ring includes substituted or unsubstituted aromatic heterocyclic rings, and the substituents include but are not limited to C 1-10 of alkyl.

[0054] Unless otherwise indicated, the term "halogen" or "halogen element" by itself or as part of another substituent refers to a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is meant to include monohaloalkyl and polyhaloalkyl. For example, "haloC 1-4 The term "alkyl" refers to, but is not limited to, trifluoromethyl, 2,2,2-trifluoromethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0055] Optical isomers, diastereomers, geometric isomers and tautomers: Some compounds of Formula I may contain one or more ring systems and may therefore exist as cis and trans isomers. The present invention is intended to encompass all such cis and trans isomers. Olefinic double bonds are included and, unless otherwise specified, are meant to include both E and Z geometric isomers.

[0056] Any enantiomer of a compound of formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

[0057] In addition, the compounds of formula I may also include a series of stable isotope labeled analogs. For example, one or more protons in the compounds of formula I may be replaced by deuterium atoms to provide deuterated analogs with improved pharmacological activity.

[0058] "Pharmaceutically acceptable salts" refer to acid or base salts of the compounds of the invention which have the desired pharmacological activity and are neither biologically desirable nor otherwise desirable. Salts can be formed with acids including, but not limited to, acetic acid, adipic acid, benzoate, citric acid, camphoric acid, camphorsulfonate, dichloroate, dodecyl sulfate, ethanesulfonate, fumarate, glucose heptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, oxalate.

[0059] The term "disorder mediated by PKMYT1 activity" refers to any disease or other deleterious condition in which PKMYT1 or mutants thereof are known to play a role.

[0060] By means of the above technical solution, the present invention has at least the following advantages:

[0061] The present invention provides a novel class of amine derivatives with PKMYT1 inhibitory activity. The compounds have high PKMYT1 inhibitory activity and can be used to treat diseases mediated by PKMYT1 activity, such as solid tumors, blood tumors, DNA damage response cancers, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a graph showing the changes in tumor volume of mice bearing human breast cancer HCC156 cells in different treatment groups over time;

[0063] Figure 2 This is a graph showing the changes in body weight of mice bearing human breast cancer HCC156 cells in different treatment groups over time. DETAILED DESCRIPTION

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0065] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0066] In the following examples of the present application, the synthesis methods of the reaction raw materials or reaction intermediates used are as follows:

[0067] (1) Preparation of intermediate arylamine A1:

[0068] The compound is prepared by the synthesis method shown in the following reaction scheme 1:

[0069]

[0070] Iron powder (0.44 g, 7.9 mmol) and FeBr3 (0.23 g, 0.8 mmol) were added to a solution of compound 1 (3.0 g, 19.8 mmol) in DCM (30 mL) at room temperature, and then Br2 (1.2 mL, 23.8 mmol) was slowly added dropwise to the reaction solution. After the addition was complete, the reaction was stirred for 3 h until TLC showed that compound 1 was completely reacted. The reaction mixture was diluted with ice water, extracted with DCM (20 mL x 3), washed with water and brine, and the organic phase was dried over anhydrous Na2SO4 and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 2 (4.3 g, yield: 94%). 1 H NMR (400MHz, CDCl3) δ7.54(d,1H),7.00(d,1H),2.33(s,3H),2.24(s,3H).

[0071] CuBr (0.54 g, 3.7 mmol) and MeONa (3.0 g, 56.1 mmol) were added to a DMF (30 mL) solution of compound 2 (4.3 g, 18.7 mmol) in sequence. The reaction mixture was slowly heated to 95 ° C and stirred for 6 h until compound 2 showed complete reaction on TLC. The reaction mixture was cooled to room temperature, diluted with EA and saturated NH4Cl, then extracted with EA (20 mL x 3), washed with water and brine, and the organic phase was dried over anhydrous Na2SO4 and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 3 (3.0 g, yield: 90%). 1 HNMR(400MHz, CDCl3) δ7.06(d,1H),6.83(d,1H),3.84(s,3H),2.22(s,3H),2.13(s,3H).

[0072] NH4CI (4.4 g, 82.7 mmol) and Fe (4.6 g, 82.7 mmol) were added to a mixed solution of EtOH (30 mL) and water (10 mL) of compound 3 (3.0 g, 16.6 mmol). The mixture was heated to 85°C for 4 h until TLC showed that compound 3 was completely reacted. The mixture was cooled to room temperature and filtered to remove most of the filtrate. The resulting mixture was extracted with EA (20 mL x 3). The organic phases were combined, washed with water and brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude product which was purified by silica gel column chromatography to obtain compound A1 (2.1 g, yield: 85%). 1H NMR (400MHz, CDCl3) δ6.89(d,1H),6.31(d,1H),3.80(s,3H),3.62(br,2H),2.15(s,3H),2.08(s,3H).

[0073] (2) Preparation of intermediate arylamine A2:

[0074] The compound is prepared by the synthesis method shown in the following reaction scheme 2:

[0075]

[0076] Under ice bath, Fe powder (150 mg, 27 mmol), FeBr3 (800 mg, 2.7 mmol) were added to a DCM (200 mL) solution of compound 3 (12 g, 67 mmol) in sequence, and then bromine (5 mL, 81 mmol) was added dropwise. After reacting for 0.5 h, the mixture was heated to room temperature and stirred in the dark for 1 h until the reaction of compound 3 was complete. The obtained solution was poured into water and washed with sodium thiosulfate until the red color disappeared. The aqueous layer was extracted with DCM (3×20 mL), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated to obtain white solid compound 4 (11 g, yield: 91%).

[0077] Under nitrogen protection, Pd2(dba)3 (1.1 g, 1.23 mmol), BINAP (767 mg, 1.23 mmol), and NaOtBu (3.5 g, 36.9 mmol) were added to a toluene (50 mL) solution of compound 4 (3 g, 12.3 mmol) and compound 5 (2.68 g, 14.8 mmol) and the reaction solution was heated to 120°C for 2 h until the reaction of compound 4 was complete. The mixture was cooled to room temperature, extracted with EA, washed with water, and the organic phase was dried, concentrated, and purified by column to obtain compound 6 (1.1 g, yield: 36%). 1 H NMR (400MHz, CDCl3) δ7.83-7.78(m,2H),7.51-7.46(m,2H),7.45-7.39(m,2H),7.34-7.31(m,2H) ,7.13-7.08(m,2H),6.02(s,1H),3.51(s,3H),2.06(s,3H),2.01(s,3H).LCMS(ESI):361.55[M+H] + .

[0078] To a solution of compound 6 (1.1 g, 3.1 mmol) in dioxane (10 mL) was added 10 mL of water and 10 mL of HCl. The reaction solution was reacted at room temperature for 1 h until compound 6 was completely reacted. The mixture was extracted with EA, washed with water, and the organic phase was dried, concentrated, and purified by column to obtain compound 7 (450 mg, yield 80%). 1 H NMR (400MHz, CDCl3) δ6.29(s,1H),3.79(s,3H),2.01(s,3H),1.97(s,3H).

[0079] Under ice bath, to a mixed solution of compound 7 (3.0 g, 0.015 mol) in HBF4 (25 mL) and H2O (25 mL) was slowly added dropwise an aqueous solution of NaNO2 (2.1 g, 0.031 mol), and the temperature of the reaction solution was kept below 5° C. During the addition, the reaction was stirred for 30 minutes, and then the mixture was filtered and washed with ice water (15 mL), ice-cold ethanol (15 mL) and ether (15 mL). The tetrafluoroborate obtained was purified by column to obtain compound 8 (1.2 g, yield: 39%). 1 H NMR (400MHz, CDCl3) δ6.84(s,1H),3.84(s,3H),2.22(s,3H),2.14(s,3H).

[0080] Iron powder (99 mg, 1.77 mmol) was added to a mixed solution of compound 8 (50 mg, 0.25 mmol) and NH4CI (189 mg, 3.5 mmol) in ethanol (5 mL) and water (2 mL). The reaction mixture was heated at 78°C for 2 h until compound 8 was completely reacted. The reaction mixture was filtered and the filter cake was washed with hot ethanol. The filtrate was dried and concentrated and then purified by column to obtain compound A2 (15 mg, yield: 36%). 1 H NMR (400MHz, CDCI3) δ6.12(d,1H), 3.76(s,3H), 2.03(d,3H), 2.01(d,3H).

[0081] (3) Preparation of intermediate arylamine A3:

[0082] The compound is prepared by the synthesis method shown in the following reaction scheme 3:

[0083]

[0084] Under ice bath, NaNO2 (3.5 g, 0.051 mol) was added in batches to a mixed solution of compound 7 (5.0 g, 0.051 mol) in AcOH (5 mL), CDCl3 (3 mL) and D2O (1 mL). The reaction mixture was stirred at room temperature for 3 h until TLC showed that the reaction of compound 7 was complete. It was extracted with DCM (30 mL x 3), washed with water and brine, and the organic phases were combined, dried over anhydrous Na2SO4 and concentrated to obtain a crude compound which was purified by silica gel column chromatography to give compound 9 (2.3 g, yield: 50%). 1 H NMR (400MHz, CDCl3) δ6.84(s,1H),3.84(s,3H),2.22(s,3H),2.13(s,3H).

[0085] To a mixed solution of compound 9 (2.3 g, 0.013 mol) in ether (20 mL) and water (4 mL), NH4CI (4.0 g, 0.076 mol) and iron powder (4.2 g, 0.076 mol) were added. The reaction mixture was heated at 78°C for 2 h until compound 9 was completely reacted, the reaction mixture was filtered, the filter cake was washed with hot ethanol, the filtrate was dried and concentrated, and the crude product was purified by silica gel column chromatography to obtain compound A3 (1.5 g, yield: 78%). 1 HNMR (400MHz, CDCl3) δ6.30(s,1H),3.79(s,3H),2.14(s,3H),2.07(s,3H).

[0086] Example 1

[0087] 2-Amino-1-(3-fluoro-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-1)

[0088] The compound is prepared according to the synthetic method shown in the following reaction scheme 4:

[0089]

[0090] The specific preparation process is as follows:

[0091] H2SO4 (3.6mL, 67.1mmol) was slowly added to water (30mL), cooled to room temperature, and then compound 10 (3.0g, 14.9mmol) was added in batches. The reaction solution was cooled to 0℃-5℃ with an ice-water bath. Under strong stirring, an aqueous solution (3mL) of NaNO2 (1.3g, 18.7mmol) was slowly added dropwise. The ice-water bath was removed, and the reaction solution was slowly heated to room temperature for 1h, and then an aqueous solution (5mL) of NaOH (4.6g, 114.1mmol) was added dropwise, keeping the temperature below 20℃. The pH was adjusted to 7 with saturated NaHCO3, extracted with DCM (20mL x 3), washed with water and brine, and the organic phases were combined, dried and concentrated with anhydrous Na2SO4. The crude compound obtained was purified by silica gel column chromatography to obtain compound 11 (3.0g, yield: 97%). 1 H NMR (400MHz, CDCl3) δ13.36(s,1H),7.62(s,1H),2.31(s,3H),2.04(s,3H).

[0092] Under nitrogen protection, a solution of POBr3 (6.4 g, 22.3 mmol) in Tol (10 mL) was added dropwise to a mixed solution of compound 11 (3.0 g, 14.8 mmol) in DMF (9 mL) and toluene (15 mL). The reaction solution was stirred at 90 ° C overnight. The mixture was cooled to room temperature and slowly added into water (50 mL). The organic phase was extracted with EA (50 mL x 3), washed with water and brine, and dried over anhydrous Na2SO4. The crude compound obtained by concentration was purified by silica gel column chromatography to obtain compound 12 (3.3 g, yield: 84%). 1 H NMR (400MHz, CDCl3) δ7.60(s,1H),2.43(s,3H),2.22(s,3H).

[0093] Under nitrogen protection, the reaction solution of compound 12 (0.78 g, 2.96 mmol) and compound A2 (0.5 g, 2.96 mmol), Pd2(dba)3 (0.27 g, 0.30 mmol), Xantphos (0.17 g, 0.30 mmol), and Cs2CO3 (2.4 g, 7.4 mmol) in DME (5 mL) was stirred and refluxed for 4 h until TLC showed that compound 12 was completely reacted. The mixture was cooled to room temperature, extracted with EA (8 mL x 3), washed with water and brine, and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated to obtain a crude compound, which was purified by silica gel column chromatography to obtain compound 13 (0.58 g, yield: 86%). 1H NMR (400MHz, CDCl3) δ7.44(s,1H),7.05(d,1H),6.72(d,1H),6.19(s,1H),3.84(s,3H),2.18(s,3H),2.17(s.3H),2.12(s,3H),2.10(s,3H).

[0094] Under nitrogen protection, NaOtBu (0.47 g, 4.9 mmol) was added to a DME (5 mL) solution of malononitrile (0.22 g, 3.3 mmol) three times. The reaction mixture was stirred at room temperature for 30 min, then compound 13 (0.58 g, 1.6 mmol) and Pd (dppf) Cl2 (0.12 g, 0.16 mmol) were added, and the reaction solution was refluxed overnight. The reaction mixture was cooled to room temperature, extracted with EA (5 mL x 3), and the combined organic phases were washed with water and brine, and the crude compound obtained by concentrating the combined organic phases after drying with anhydrous Na2SO4 was purified by silica gel column chromatography to obtain compound 14 (470 mg, yield: 84%). 1 H NMR (400MHz, CDCl3) δ7.51(s,1H),6.78(d,1H),4.52(s,2H),3.60(s,3H),2.38(s,3H),2.32(s.3H),1.81(s,3H),1.78(s,3H).

[0095] At room temperature, 0.2 mL of water and 0.5 mL of sulfuric acid were slowly added to methanesulfonic acid (2.7 mL, 41.7 mmol), and then compound 14 (470 mg, 1.39 mmol) was added in batches, keeping the reaction temperature below 40° C. The resulting solution was stirred at room temperature until TLC showed that compound 14 was completely reacted, the pH was adjusted to 7 with saturated aqueous NaOH solution, the reaction mixture was extracted with EA (8 mL×3), washed with water and brine, the organic phases were combined, dried over anhydrous Na2SO4, and concentrated to obtain a crude compound, which was purified by silica gel column chromatography to obtain compound 15 (410 mg, yield: 83%). 1 H NMR (400MHz, DMSO) δ7.86(s,1H),7.09(d,1H),6.88(s,2H),6.69(s,2H),3.86(s,3H),2.27(s,3H),2.24(s.3H),1.70(s,3H),1.65(s,3H).

[0096] At 0°C, BBr3 (0.5 ml, 4.72 mmol) was added dropwise to a DCM (8 mL) solution of compound 15 (410 mg, 1.15 mmol). The reaction mixture was stirred at 50°C for 1 h until TLC showed that compound 15 was completely reacted. The solvent was removed, and the residue was dissolved in MeOH and concentrated to dryness again. The solid was washed with DCM:MeOH=10:1, and the organic phase was washed with a saturated aqueous NaHCO3 solution and then dried and concentrated to give compound I-1 (355 mg, yield: 90%). 1 H NMR(400MHz,DMSO)δ9.98(s,1H),7.86(s,1H),6.86(s,2H),6.84(d,1H),6.67 (s,2H),2.27(s,3H),2.26(s.3H),1.66(s,3H),1.62(s,3H).MS:[M+1]:343.2.

[0097] Example 2

[0098] 2-Amino-1-(3-deuterium-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-2)

[0099] The compound is prepared according to the synthetic method shown in the following reaction scheme 5:

[0100]

[0101] Under nitrogen protection, a mixture of compound 12 (2.2 g, 8.5 mmol) and compound A3 (1.3 g, 8.5 mmol), Pd2(dba)3 (0.8 g, 0.85 mmol), Xantphos (0.5 g, 0.33 mmol), and Cs2CO3 (7.0 g, 21.4 mol) in DME (15 mL) was heated under reflux for 13 h until TLC showed that compound 12 was completely reacted. The reaction solution was cooled to room temperature and extracted with EA (10 mL x 3), the organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude compound which was purified by silica gel column chromatography to obtain compound 16 (2.0 g, yield: 71%). 1H NMR (400MHz, CDCl3) δ7.43(s,1H),6.72(s,1H),6.18(s,1H),3.83(s,3H),2.17(s,3H),2.16(s.3H),2.11(s,3H),2.10(s,3H).

[0102] Under nitrogen protection, NaOtBu (860 mg, 9.0 mmol) was added three times to a DME (10 mL) solution of malononitrile (394 mg, 6.0 mmol). The reaction mixture was stirred at room temperature for 30 min, then compound 16 (1.0 g, 3.0 mmol) and Pd (dppf) Cl2 (218 mg, 0.3 mmol) were added, and the reaction was refluxed overnight. The reaction mixture was cooled to room temperature, extracted with EA (8 mL x3), the organic phase was washed with water and brine, the organic phases were combined, dried over anhydrous Na2SO4, and then concentrated to obtain the crude compound obtained by the reaction, and purified by silica gel column chromatography to obtain compound 17 (420 mg, yield: 44%). 1 H NMR (400MHz, CDCl3) δ7.49(s,1H),6.95(d,1H),4.52(s,2H),3.87(s,3H),2.37(s,3H),2.32(s.3H),1.91(s,3H),1.84(s,3H).

[0103] At room temperature, sulfuric acid (0.5 mL) and water (0.2 mL) were slowly added to methanesulfonic acid (2.5 mL, 39.2 mmol), and then compound 17 (420 mg, 1.3 mmol) was added in batches, and the reaction temperature was kept below 40° C. The resulting solution was stirred at room temperature for 90 min until TLC showed that compound 17 was completely reacted, and the pH was adjusted to 7 with saturated aqueous NaOH solution. The reaction mixture was extracted with EA (8 mL x 3), and the organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude compound which was purified by silica gel column chromatography to obtain compound 18 (430 mg, yield: 97%). 1 H NMR (400MHz, DMSO) δ7.83(s,1H),7.08(s,1H),6.75(s,2H),6.65(s,2H),3.85(s,3H),2.27(s,3H),2.24(s.3H),1.80(s,3H),1.70(s,3H).

[0104] At 0°C, BBr3 (0.5 mL, 5.20 mmol) was added dropwise to a DCM (8 mL) solution of compound 18 (430 mg, 1.27 mmol). The reaction mixture was then stirred at room temperature for 1 h until TLC showed that compound 18 was completely reacted. The solvent was removed, and the residue was dissolved in MeOH and concentrated to dryness again. The solid was washed with DCM:MeOH=10:1, and the organic phase was washed with a saturated NaHCO3 solution, dried over anhydrous Na2SO4, and concentrated to obtain compound I-2 (395 mg, yield: 96%). 1H NMR (400MHz, DMSO) δ9.49(s,1H),7.82(s,1H),6.90(s,1H),6.70(s,2H),6.63(s,2H),2.26(s,3H),2.24(s.3H),1.74(s,3H),1.66(s,3H). MS:[M+1]:326.2.

[0105] Example 3

[0106] 2-Amino-1-(3-fluoro-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-3)

[0107]

[0108] Compound I-3 was obtained from compound I-1 by chiral SFC separation (instrument: Waters Prep100 SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30 x 250 mm, 5 pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70 mL / min).

[0109] Peak 1 (retention time 2.308 min, 99.3%), MS: [M+1]: 343.1.

[0110] Example 4

[0111] 2-Amino-1-(3-fluoro-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-4)

[0112]

[0113] Compound I-4 was obtained from compound I-1 by chiral SFC separation (instrument: Waters Prep100 SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30 x 250 mm, 5 pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70 mL / min).

[0114] Peak 2 (retention time 3.869 min, 98.9%), MS: [M+1]: 343.1.

[0115] Example 5

[0116] 2-Amino-1-(3-deuterium-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-5)

[0117]

[0118] Compound I-5 was obtained from compound I-2 by chiral SFC separation (instrument: Waters Prep100 SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30 x 250 mm, 5 pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70 mL / min).

[0119] Peak 1 (retention time 2.523 min, 99.5%), MS: [M+1]: 326.2.

[0120] Example 6

[0121] 2-Amino-1-(3-deuterium-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (I-6)

[0122]

[0123] Compound I-6 was obtained from compound I-2 by chiral SFC separation (instrument: Waters Prep100 SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30 x 250 mm, 5 pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70 mL / min).

[0124] Peak 2 (retention time 3.987 min, 100%), MS: [M+1]: 326.2.

[0125]

[0126] Example 7

[0127] 2-Amino-1-(3-fluoro-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[3,2-b]pyridine-3-carboxamide (I-7)

[0128]

[0129] Prepared from compound 12 and intermediate A2 according to the synthetic method shown in reaction scheme 6, MS: [M+1]: 343.2.

[0130] Example 8

[0131] 2-Amino-1-(3-deuterium-5-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[3,2-b]pyridine-3-carboxamide (I-8)

[0132]

[0133] Prepared from compound 12 and intermediate A3 according to the synthetic method shown in reaction scheme 6, MS: [M+1]: 343.2.

[0134] Example 9

[0135] 2-Amino-1-(3-hydroxy-2,6-dimethylphenyl)-5,6,7,8-tetrahydropyrrolo[3,2-e]pyrido[2,3-b]pyridine-3-carboxamide (I-9)

[0136] The compound was prepared according to the synthetic method shown in the following reaction scheme 7:

[0137]

[0138] At room temperature, NBS (5.98 g, 0.033 mol) was added in batches to a solution of compound 19 (3.0 g, 0.011 mol) in DCM (30 mL), and the reaction was stirred for 3 h until TLC showed that the reaction of compound 19 was complete. The reaction mixture was diluted with ice water, extracted with DCM (20 mL x 3), and the organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 20 (2.72 g, yield: 70%). 1 H NMR (400MHz, CDCl3) δ7.58(s,1H),3.75(t,2H),2.73(t,2H),1.95-1.89(m,2H),1.53(s,9H).

[0139] Under nitrogen protection, a mixture of compound 20 (1.5 g, 0.0043 mol) and compound A1 (0.79 g, 0.0052 mol), Pd2(dba)3 (0.40 g. 0.43 mmol), Xantphos (0.25 g, 0.43 mmol), and Cs2CO3 (3.53 g, 0.011 mol) in DME (15 mL) was heated under reflux for 13 h until TLC showed that the reaction of compound 20 was complete. The mixture was cooled to room temperature and extracted with EA (5 mL x 3). The organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated to give a crude product which was purified by silica gel column chromatography to give compound 21 (1.5 g, yield: 75%). 1H NMR(400MHz, CDCl3)δ7.08(d,1H),6.75(d,1H),6.17(s,1H),5.53(s,1H),3.85(s,3H) ,3.69(t,2H),2.55(t,2H),2.12(s,3H),2.06(s.3H),1.86-1.80(m,2H),1.54(s,9H).

[0140] Under nitrogen protection, NaOtBu (0.75 g, 0.0078 mol) was added in batches to a solution of malononitrile (0.34 g, 0.0052 mol) in DME (8 mL) and the mixture was stirred at room temperature for 30 minutes. Compound 21 (1.2 g, 0.0026 mol) and Pd(dppf)Cl2 (0.19 g, 0.26 mmol) were added and the reaction solution was stirred and refluxed overnight. The reaction solution was cooled to room temperature and extracted with EA (5 mL x 3). The organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude compound which was purified by silica gel column chromatography to obtain compound 22 (1.02 g, yield: 88%). 1 H NMR(400MHz, CDCl3)δ7.20(d,1H),6.95(d,1H),6.58(s,1H),4.84(s,2H),3.88 (s,3H),3.74(t,2H),2.67(t,2H),1.92-1.88(m,5H),1.82(s.3H),1.58(s,9H).

[0141] Sulfuric acid (0.5 mL) and water (0.2 mL) were slowly added to methanesulfonic acid (4.4 mL, 0.068 mol) at room temperature, and then compound 22 (1.02 mg, 0.0023 mol) was added in batches, keeping the reaction temperature below 40° C. The resulting solution was stirred at room temperature for 1.5 h until TLC showed that compound 22 was completely reacted, and the pH was adjusted to 7 with NaOH, extracted with EA (8 mL x 3), washed with water and brine, and the organic phase was dried over anhydrous Na2SO4 and concentrated to obtain a crude compound, which was purified by silica gel column chromatography to obtain compound 23 (0.71 g, yield: 86%). 1 H NMR(400MHz, CDCl3)δ7.36(s,1H),8.19(s,1H),7.17(d,1H),6.91(d,1H),6.46(s,1H),5.58(s,2H) ,5.25(s,1H),4.56(s,1H),3.87(s,3H),3.39(t,2H),2.68(s,3H),1.93-1.87(m,5H),1.85(s,3H).

[0142] Under ice bath, BBr3 (0.7 mL, 0.0078 mol) was added dropwise to a DCM (10 mL) solution of compound 23 (0.71 g, 0.0019 mol). After addition, the reaction mixture was stirred at room temperature for 1 h until TLC showed that compound 23 was completely reacted. The solvent was removed, and the residue was dissolved in MeOH and concentrated to dryness again. The solid was washed with DCM:MeOH=10:1, and the organic phase was washed with saturated NaHCO3 solution, dried over anhydrous Na2SO4, and concentrated to obtain compound I-9 (0.58 g, yield: 85%). 1 H NMR (400MHz, DMSO) δ9.54(s,1H),7.90(s,1H),7.06(d,1H),6.90(d,1H),6.83(s,1H),6.44(s,2H),6. 33(s,1H),5.96(s,1H),3.17(t,2H),2.61(t,2H),1.79-1.73(m,5H),1.70(s,3H).MS:[M+1]:352.65.

[0143] Example 10

[0144] 2-Amino-1-(3-hydroxy-2,6-dimethylphenyl)-5,6,7,8-tetrahydropyrrolo[3,2-e]pyrido[2,3-b]pyridine-3-carboxamide (I-10)

[0145]

[0146] Compound I-10 was obtained from compound I-9 by chiral SFC separation (instrument: Waters Prep100SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30x 250mm, 5pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70mL / min).

[0147] Peak 1 (retention time 0.591 min, 99.1%), MS: [M+1]: 352.4.

[0148] Embodiment 11

[0149] 2-Amino-1-(3-hydroxy-2,6-dimethylphenyl)-5,6,7,8-tetrahydropyrrolo[3,2-e]pyrido[2,3-b]pyridine-3-carboxamide (I-11)

[0150]

[0151] Compound I-11 was obtained from compound I-9 by chiral SFC separation. Chiral SFC separation (instrument: Waters Prep100SFC-MS; chromatographic column: Phenomenex Lux cellulose-2, 30x 250mm, 5pm; conditions: 50% ACN / EtOH 1:1, 50% CO2 isobaric; flow rate: 70mL / min)

[0152] Peak 2 (retention time 1.387 min, 99.5%), MS: [M+1]: 352.4.

[0153] Test Example 1

[0154] The in vitro inhibitory activity test of PKMYT1 was performed on some of the compounds synthesized in the above examples, with RP-6306 and PD0166285 as control samples. The specific operation is as follows:

[0155] (1) Dissolve the compound in DMSO solution to prepare a 20 mM compound stock solution and store it in a nitrogen cabinet away from light;

[0156] (2) Dilute the 20 mM compound stock solution to a 1 μM compound working solution. The test concentration of the test compound is 1000 nM, 3-fold dilution, 10 concentrations;

[0157] (3) Add 0.1uL of compound test solution to each well of the 384-well plate for enzyme test, then add 5uL of enzyme working solution, centrifuge at 1000rpm for 1 minute, and then incubate the 384-well plate at 25℃ for 10 minutes. Next, add 5uL of substrate and ATP working solution to each well, incubate at 25℃ for 3 hours, then add 5uL of ADP-Glo ​​reagent, centrifuge at 1000rpm for 1 minute, and then incubate the 384-well plate at 25℃ for 40 minutes. Finally, add 10uL of detection solution to each well, centrifuge at 1000rpm for 1 minute, and then incubate the 384-well plate at 25℃ for 40 minutes. Use BMG to read the luminescent signal for subsequent parameter calculations. Calculation formula: Inhibition rate (% Inh) = (maximum signal-compound signal) / (maximum signal-minimum signal)×100. Minimum signal: the mean of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Maximum signal: the mean of the positive control wells, representing the conversion rate reading of the wells without compound inhibition;

[0158] (4) Fitting the dose-effect curve: The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve to obtain the IC value of each compound on the enzyme activity. 50The value is as follows:

[0159] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)×HillSlope))

[0160] The test results are shown in Table 1 below:

[0161] Table 1 In vitro inhibition test IC of different compounds on PKMYT1 50 Test Results

[0162]

[0163]

[0164] According to the above PKMYT1 in vitro inhibition assay IC 50 The test results show that the compounds provided by the present invention exhibit high inhibitory activity against PKMYT1, among which compounds I-1, I-2, I-3, I-5, etc. have inhibitory activity similar to or even better than that of RP-6306.

[0165] Test Example 2

[0166] The inhibitory activity of some of the compounds synthesized in the above examples and RP-6306 on HCC1569 cells was studied, and the specific operation was as follows:

[0167] Cell Viability Test Protocol

[0168] (1) Plating: Digest the cells with 0.25% Trypsin-EDTA, resuspend the cells and count them using an automated cell counter. Dilute the cell suspension to the desired density based on the seeding density. Add 200 μL of cells to each well and culture overnight at 37°C, 5% CO2.

[0169] (2) Compound preparation: Prepare the compound into a 200-fold final solution with DMSO. Add 3 μL of the 200-fold final solution to 197 μL of culture medium to prepare a 3-fold final solution. Add 50 μL of the 3-fold final solution to the well plate and culture at 37°C, 5% CO2 for 7 days.

[0170] (3) Detection: Equilibrate the test plate to room temperature. Add 40 μL of Reagent, shake for 2 minutes, and incubate at room temperature for 60 minutes. Detect with Envision.

[0171] Data analysis

[0172] (1) Calculation of IC using GraphPad Prism 5 50 .

[0173] (2)%Inh=(Max signal-Compound signal) / (Max signal-Min signal)×100.

[0174] (3) Max signal is when only DMSO is added without adding any compound.

[0175] (4) Min signal is when only culture medium is added to the entire well plate.

[0176] Table 2 IC of some compounds in cell tests 50 (Unit: nM)

[0177]

[0178]

[0179] As shown in Table 2, compared with RP-6306, the compounds provided by the present invention exhibit higher inhibitory activity against HCC1569 cells.

[0180] Test Example 3

[0181] The present invention further studies the in vivo PK activity of such compounds, and the specific operation is as follows:

[0182] Prepare 6 male SD rats aged 6-8 weeks, weighing 180-300g, and adapt to culture for 3-7 days. Use 10% DMSO + 90% (30% HP-β-CD) as the solvent, and adjust the concentration of the test compound to 0.5mg / mL and 1mg / mL, respectively. Divide the animals into 3 intravenous injection groups and 3 oral gavage groups, and give them 1mg / kg and 10mg / kg doses, respectively. The intravenous injection group was administered with 0.5mg / mL of the test compound, and the administration volume was 2mL / kg; the oral gavage group was administered with 1mg / mL of the test compound, and the administration volume was 10mL / kg. Blood was collected from the jugular vein of rats in the intravenous injection group at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h and 24h after administration; blood was collected from the jugular vein of rats in the oral gavage group at 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h and 24h. All blood samples were transferred to plastic centrifuge tubes containing anticoagulants, centrifuged at 4000g, 4℃ for 5min, and the supernatant was transferred to a microcentrifuge tube without anticoagulants. The plasma was stored in a refrigerator at -75±15℃, and the drug concentration in plasma was determined by LC-MS / MS. After obtaining the drug concentration-time curve, the corresponding pharmacokinetic parameters were calculated using pharmacokinetic software.

[0183] Table 3-1 Pharmacokinetic parameters of different compounds in SD rats (n=3, intravenous injection of 1 mg / kg)

[0184]

[0185] Table 3-2 Pharmacokinetic parameters of different compounds in SD rats (n=3, oral administration 10 mg / kg)

[0186]

[0187]

[0188] Note: Values ​​= mean ± SD.

[0189] The PK test results show that the compounds provided by the present invention have good bioavailability and high exposure, and good drugability.

[0190] Test Example 4

[0191] The present invention further conducts an in vivo efficacy test on the compounds in mice, and the specific operation is as follows:

[0192] NOD SCID mice were ordered from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. and assigned to this study at 6-8 weeks of age. Animal husbandry, feeding, and health conditions were in accordance with animal welfare guidelines. Human breast cancer HCC1569 cells were cultured in RPMI-1640 medium + 10% FBS medium and passaged at least 3 times before inoculation. Each mouse was inoculated with approximately 5×10 6 Human breast cancer HCC1569 cells were collected in a sterile PBS solution, and the cell suspension was added with matrix gel at a volume ratio of 1:1. The inoculation volume was about 200 μL. NOD SCID mice were anesthetized with 3-4% isoflurane in advance. When the tumor grew to an average of about 100-200 mm 3 At about 24 hours, 24 mice with appropriate tumor size were randomly divided into 4 groups according to tumor size and body weight, with 6 mice in each group, namely Vehicle (G1 group), I-3 20mg / kg (D0~D14) / 15mg / kg (D15~D27) (G2 group), I-57mg / kg (G3 group), I-5 20mg / kg (D0~D14 / D25~27) / 15mg / kg (D15~D24)) (G4 group), and all 4 groups were orally administered for 28 days. The results of tumor volume inhibition rate are shown in the table.

[0193] Tumor volume (V) was calculated as follows: V = (length × width 2 ) / 2.

[0194] Tumor inhibition rate (TGI) = [1-(TV t -TV initial ) / (CV t -CVinitial )]*100%; among which,

[0195] TV t :Tumor volume of treatment group on Day 27, TV initial : Tumor volume of treatment group on Day 0; CV t :Day 27 control group tumor volume, CV initial : Tumor volume of the control group on Day 0.

[0196] Table 4 Effects of different compounds on tumor proliferation rate in mice bearing human breast cancer HCC156 cells

[0197]

[0198]

[0199] Note: There were 6 animals in each group. The data of tumor volume (mm3) are mean ± SD. The dosing method of mice in each group was: pobi d*28D (oral administration twice a day, for 28 days); Day 27 means the 28th day of dosing.

[0200] Combination Figure 1 As shown in Table 4, I-3 and I-5 had significant inhibitory effects on tumor growth in mice bearing human breast cancer HCC156 cells, among which the I-5 high-dose {20 mg / kg (D0~D14 / D25~27) / 15 mg / kg (D15~D24))} group had better tumor inhibition effect than the I-5 low-dose group (7 mg / kg).

[0201] Depend on Figure 2 It can be seen that during the drug administration process of the human breast cancer HCC156 cell tumor-bearing mouse model, the body weight of the mice in the I-3 dose group decreased rapidly within one week of administration, and the body weight gradually stabilized after about 2 weeks of administration, with a body weight decrease of about 10% to 15%; the mice in the I-57 mg / kg dose group did not show obvious weight loss, while the mice in the I-520 mg / kg (D0 to D14 / D25 to 27) / 15 mg / kg (D15 to D24) dose group had a slight decrease in body weight and were in good condition.

[0202] In summary, the compounds provided by the present invention have high inhibitory activity against PKMYT1 and good drugability, and have extremely high application value in the preparation of drugs for preventing and / or treating diseases mediated by PKMYT1 activity in vivo.

[0203] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A compound of formula I or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph, in, X, Y, and Z are independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl; Or, Y, X and / or Z and the connected carbon atoms form a cyclic structure, wherein the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring, and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O; U is N or C. When U is N, X does not exist. W is N or CR 1 , where R 1 is selected from hydrogen, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 One of cycloalkyl, aryl, heteroaryl, alkenyl, and alkynyl; G1 and G2 are independently selected from one of hydrogen, deuterium and fluorine.

2. The compound according to claim 1, characterized in that The compound is a compound represented by Formula Ia to Ie or an isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph thereof, in, In Formula Ia, X, Y, and Z are independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl; In Formula Ib, X is selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl; Y and Z and the connected carbon atoms form a cyclic structure, the cyclic structure is selected from one or more of alicyclic rings, aromatic heterocyclic rings, and alicyclic heterocyclic rings, and the heteroatoms in the aromatic heterocyclic rings and alicyclic heterocyclic rings are N and / or O; In Formula Ic, Y, X and the connected carbon atoms form a cyclic structure, and the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring, and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O to form a cyclic structure; Z is selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl; In Formula Id, Y and Z are independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Cycloalkyl; In Formula Ie, Y and Z and the connected carbon atoms form a cyclic structure, and the cyclic structure is selected from one or more of an alicyclic ring, an aromatic heterocyclic ring, and an alicyclic heterocyclic ring, and the heteroatoms in the aromatic heterocyclic ring and the alicyclic heterocyclic ring are N and / or O to form a cyclic structure.

3. The compound of formula I according to claim 1 or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph, characterized in that: X, Y, and Z are independently selected from one of hydrogen, chlorine, fluorine, cyano, methyl, ethyl, propyl, fluoromethyl, trifluoromethyl, and deuterated methyl; or, Y, X and / or Z and the connected carbon atom form a substituted or unsubstituted C 3-10 Alicyclic, substituted or unsubstituted C 3-10 Aromatic heterocyclic ring, substituted or unsubstituted C 3-10 Aliphatic heterocycle.

4. The compound of formula I according to claim 1 or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph, characterized in that: The compound represented by formula I is a compound represented by the following structural formulas I-1 to I-50:

5. A pharmaceutical composition, characterized in that The invention relates to a compound represented by formula (I) as described in any one of claims 1 to 4, and one or more of its isotopic forms, stereoisomers, tautomers, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, hydrates, prodrugs and polymorphs.

6. Use of a compound of formula (I) according to any one of claims 1 to 4 or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug, polymorph, or the pharmaceutical composition according to claim 5 in the preparation of a PKMYT1 inhibitor.

7. The use according to claim 6, characterized in that: The PKMYT1 inhibitor is administered orally, parenterally, intravenously or transdermally.

8. Use of a compound of formula (I) according to any one of claims 1 to 4 or its isotopic form, stereoisomer, tautomer, pharmaceutically acceptable salt, pharmaceutically acceptable solvate, hydrate, prodrug and polymorph, and the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating a disease mediated by PKMYT1 activity.

9. The use according to claim 8, characterized in that: Conditions mediated by PKMYT1 activity include solid tumors, hematological tumors, and DNA damage response cancers; the DNA damage response cancers include, but are not limited to, lung cancer, breast cancer, endometrial cancer, gastric cancer, ovarian cancer, and prostate cancer.

10. The use according to claim 8, characterized in that: The drug is administered orally, parenterally, intravenously or transdermally.

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